biomedicines

Review Mechanisms Underlying Resistance to FLT3 Inhibitors in

Motoki Eguchi 1, Yosuke Minami 1,*, Ayumi Kuzume 1,2 and SungGi Chi 1

1 Department of Hematology, National Cancer Center Hospital East, Kashiwa 277-8577, Japan; [email protected] (M.E.); [email protected] (A.K.); [email protected] (S.C.) 2 Division of Hematology/Oncology, Department of Internal Medicine, Kameda Medical Center, Kamogawa 296-8602, Japan * Correspondence: [email protected]; Tel.: +81-4-7133-1111; Fax: +81-7133-6502

 Received: 11 June 2020; Accepted: 16 July 2020; Published: 24 July 2020 

Abstract: FLT3-ITD and FLT3-TKD mutations were observed in approximately 20 and 10% of acute myeloid leukemia (AML) cases, respectively. FLT3 inhibitors such as , and quizartinib show excellent response rates in patients with FLT3-mutated AML, but its duration of response may not be sufficient yet. The majority of cases gain secondary resistance either by on-target and off-target abnormalities. On-target mutations (i.e., FLT3-TKD) such as D835Y keep the TK domain in its active form, abrogating pharmacodynamics of type II FLT3 inhibitors (e.g., midostaurin and quizartinib). Second generation type I inhibitors such as gilteritinib are consistently active against FLT3-TKD as well as FLT3-ITD. However, a “gatekeeper” mutation F691L shows universal resistance to all currently available FLT3 inhibitors. Off-target abnormalities are consisted with a variety of somatic mutations such as NRAS, AXL and PIM1 that bypass or reinforce FLT3 signaling. Off-target mutations can occur just in the primary FLT3-mutated clone or be gained by the evolution of other clones. A small number of cases show primary resistance by an FL-dependent, FGF2-dependent, and stromal CYP3A4-mediated manner. To overcome these mechanisms, the development of novel agents such as covalently-coupling FLT3 inhibitor FF-10101 and the investigation of combination therapy with different class agents are now ongoing. Along with novel agents, gene sequencing may improve clinical approaches by detecting additional targetable mutations and determining individual patterns of clonal evolution.

Keywords: acute myeloid leukemia (AML); FMS-like 3 (FLT3); quizartinib; gilteritinib

1. Introduction FMS-like tyrosine kinase 3 (FLT3) is classified as a type 3 , along with KIT, FMS, and PDGFR [1–3]. FLT3 is composed of an extracellular region consisting of five immunoglobulin-like domains, and an intracellular region consisting of a juxtamembrane (JM) domain, two tyrosine kinase (TK) domains, and a C-terminal domain. FLT3 is expressed in normal hematopoietic stem cells and progenitor cells, and is dimerized upon binding with either membrane-bound or soluble FLT3 ligands (FLs) produced by bone marrow stromal cells, which subsequently causes the phosphorylation and activation of tyrosine residues in the activation-loop (A-loop) [4,5]. Phosphorylated FLT3 activates multiple intracellular signaling pathways involved in the survival, proliferation, and differentiation of hematopoietic stem cells, such as RAS/MAPK, PI3K/Akt/mTOR, and JAK/STAT5 [6–9]. Since FLT3 is frequently expressed in leukemic cells, FL stimulation induces proliferation and inhibits apoptosis in these cells [10,11]. In 1996, an internal tandem duplication in the JM domain-encoding region of FLT3 (FLT3-ITD) was identified in acute myeloid leukemia (AML) cells [12]. Thereafter, several types of mutations, including point mutations, deletions, and insertions

Biomedicines 2020, 8, 245; doi:10.3390/biomedicines8080245 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 245 2 of 21 have been detected around the D835 residue in the TK domain (FLT3-TKD) [13]. FLT3-ITD and FLT3-TKD mutations were observed in approximately 20 and 10% of AML cases, respectively [14–16]. Although both FLT3-ITD and FLT3-TKD are gain-of-function mutations, the upregulation of STAT5 was only observed in FLT3-ITD cell lines (32D/ITD) [17]. STAT5 positively regulated Pim-1, which eventually activated mTOR and Mcl-1, which consequently conferred resistance to Akt inhibition in FLT-ITD cell lines [18]. An experiment using transgenic mice with FLT3-ITD-positive hematopoietic stem cells revealed the clear promoting effects of nuclear factors in activated T-cells (NFATC1), a family of inflammatory transcriptional factors, on FLT3-ITD-driven precursor cell expansion and resistance to FLT3 inhibitors [19]. Recent studies suggest that circulating MYBL2, encoded by the cell-cycle checkpoint gene MYBL2, is detected in AML patients with FLT3-ITD mutations and is closely related to mutant FLT3 expression as well as to tumor cell activity [20]. Unlike FLT3-ITD consistently upregulating JAK/STAT signaling, FLT3-TKD enhance SHP1 and SHP2 activity that negatively regulate JAK signaling [21,22]. This may at least partially explain why FLT3-ITD showed more potent myeloproliferative advantages than those of FLT3-TKD in a mouse model [23,24]. The dual mutation of FLT3-ITD and -TKD (FLT3-ITD-TKD) has been found in a small population. A recent study showed that FLT3-ITD-TKD has the ability to activate STAT5, resulting in Bcl-x and RAD51 upregulation that accounts for drug resistance [25]. Since FLT3 mutations are frequently detected in AML and are associated with poor prognosis, this gene is considered a promising molecular target for AML [26,27]. It has been 20 years since abnormalities in the FLT3 were first discovered, and the application of FLT3 inhibitors in clinical settings in Japan, Europe, and the United States has resulted in a paradigm shift in the treatment of FLT3-mutated AML. However, resistance to FLT3 inhibitors has also been reported concomitantly. Mechanisms of the resistance and strategies to overcome it have been vigorously studied and ever-reviewed [28–30]. Along with the comprehensive understanding of pathologic FLT3 signaling and the acquired alterations responsible for drug-resistance, non-FLT3 abnormalities that may be closely associated with leukemic clone evolution are revealing its importance, suggesting new approaches. In this review, we summarize our current understanding of resistance to FLT3 inhibitors and discuss the strategies for overcoming this issue.

2. Prognostic Impact of FLT3 Mutations FLT3-ITD mutation has been recognized as one of the major adverse prognostic factors with nearly twice the increase in hazard ratio [31]. As mentioned in the European LeukemiaNet (ELN) recommendations [27], high allelic burden (generally indicating 50% or more) of FLT3-ITD (FLT3-ITDhigh) is consistently associated with worse prognosis [32–34]. On the other hand, the low allelic frequency of FLT3-ITD (FLT3-ITDlow) concomitant with NPM1 mutation possibly leads to favorable prognosis [35], though it has been fraught with controversy [36–38]. FLT3-ITDhigh with wild type NPM1 and FLT3-ITDlow with mutated NPM1 are classified as intermediate-risk [27]. Unlike FLT3-ITD, the prognostic significance of FLT3-TKD has not been determined [32,39]. With the development of potent FLT3 inhibitors, better clinical outcomes would be expected, especially in patients with FLT3-ITDhigh. Indeed, previously untreated FLT3-ITDhigh patients who received intensive chemotherapy with , a FLT3 inhibitor, showed no significant but seemingly better relapse-free and overall survival than those with FLT3-ITDlow AML [34]. It is not fully known if the FLT3 allelic burden affects the properties in acquiring resistance to FLT3 inhibitors. However, given a certain somatic mutation will belong to a single clone, a larger proportion of mutant FLT3 allele may link to less divergent leukemic clones and vice versa, which theoretically affect drug sensitivity, relapse rates and eventually survival rates. Zhang and his colleagues graphically displayed the clonal evolutions of two individual cases; one for a single clone with a high frequency of FLT3-TKD that later relapsed with an additional mutation within the same clone and the other for complex clones not associated with first-detected FLT3-ITD mutation with low frequency [40]. The prognostic impact of FLT3 mutations and its allele frequency possibly be changed in the era of FLT3 inhibitors. Biomedicines 2020, 8, 245 3 of 21

3. Classification of FLT3 Inhibitors by Its Pharmacodynamics As first-generation FLT3 inhibitors, existing TK inhibitors such as tandutinib (CT53518), (CEP-701), (SU11248), midostaurin (PKC412), and sorafenib (BAY 43-9006), which can effectively inhibit FLT3 kinase have been studied [41–45]. Thereafter, the compounds with higher selectivity and inhibitory activity were identified. Gilteritinib (ASP2215), quizartinib (AC220), and (CP868596) were developed as second-generation FLT3 inhibitors [46–50]. These FLT3 inhibitors are roughly classified into two types (i.e., type I and type II) based on their binding mode to FLT3 molecules. The conformation of the three amino acid residues Asp–Phe–Gly (DFG) in the A-loop of the FLT3 molecule is altered in accordance with the phosphorylation status of the tyrosine residue, which leads to the formation of an active DFG-in conformation or an inactive DFG-out conformation [51–53]. Type I inhibitors bind to the ATP-binding site and its vicinity, and subsequently bind with molecules in both DFG-in and DFG-out conformations. Since the molecular homology of various TKs is high and the ATP-binding sites are highly conserved among kinases, type I inhibitors are often less selective. In contrast, type II inhibitors bind to the target kinase by utilizing the hydrophobic space that appears in the proximity of the ATP-binding site in the DFG-out conformation. Since the hydrophobic space in this structure varies significantly between various kinases, type II inhibitors are expected to be more selective than type I inhibitors and are unable to inhibit activated kinases in the DFG-in conformation. Midostaurin, gilteritinib, and crenolanib are type I inhibitors, while quizartinib and sorafenib are type II inhibitors [54]. FLT3-TKD maintains a constant DFG-in conformation owing to alterations in the TK domain, whereas FLT3-ITD can exist in both active DFG-in conformation and inactive DFG-out conformation. Therefore, while type I inhibitors inhibit both FLT3-TKD and FLT3-ITD, type II inhibitors only inhibit FLT-ITD owing to the differences in binding properties, with a few exceptions in first-generation agents (e.g., midosutaurin and sunitinib). For example, TK domain-altering D835 point mutations confer resistance to a type II second-generation inhibitor quizartinib, but not to type I gilteritinib and crenolanib [55]. However, a “gatekeeper” mutation F691L shows universal resistance to all the currently available FLT3 inhibitors [47,49,56–59]. The characteristics of the FLT3 inhibitors are summarized in Table1.

Table 1. FLT3 inhibitors.

Drug Sensitivity Agent Generation Type Selectivity IC50 (nM) ITD D835Y ITD-D835Y F691L Midostaurin (PKC412) First I Low 139 S S R R Sunitinib (SU11248) First I Low 250 S R R R Lestaurtinib (CEP701) First I Low 5 S Int S − Gilteritinib (ASP2215) Second I Moderate 1.6 S S Int R Crenolanib (CP868596) Second I Moderate 2 S S Int R Sorafenib (BAY43-9006) First II Moderate 58 S R R R Tandutinib (CT53518) First II High 100 S R − − Quizartinib (AC220) Second II High <1.0 S R R R S (sensitive) means the IC50 is less than or equal to that of FLT3-ITD. R (resistant) means more than two folds increase in IC50. Int (intermediate) remains a 1.0–2.0-folds increase. Here is the reference of selectivity [60–62], IC50 for FLT3-ITD [47,49,63–68] and drug sensitivity [47,55,69–72].

4. Current Clinical Role of FLT3 Inhibitors Among a number of tyrosine kinase inhibitors active against pathologic FLT3 signaling, gilteritinib and midostaurin are now available for the treatment of FLT3-mutated AML in most developed countries. Quizartinib is currently available only in Japan. Stone and his colleagues reported a randomized phase 3 , RATIFY, where midostaurin or placebo were added to standard therapy in patients with newly diagnosed FLT3-mutated AML [73]. The midosutaurin group showed longer survival (hazards ration (HR) 0.78) and improved event-free interval (HR 0.78) than the counterpart. Recently, the combination of midostaurin and standard therapy followed by midostaurin maintenance also showed better outcomes compared with historical controls (HR 0.58 in event-free survival) [74]. Efficacy Biomedicines 2020, 8, 245 4 of 21 of single-agent gilteritinib for relapsed/refractory FLT3-mutated AML was proved in a randomized phase 3 trial, ADMIRAL [75]. The median overall survival was significantly longer in the gilteritinib group than the conventional chemotherapy group (9.3 months vs. 5.6 months), with a higher percentage of patients who underwent allo-stem cell transplantation (SCT) (26% vs. 15%). However, the median event-free interval was less than 3 months. Similarly, the phase 3 QuANTUM-R trial showed the superiority of single-agent quizartinib over salvage chemotherapy in the same situation (HR 0.76 in overall survival) [76]. Quizartinib has also been tested in the first-line setting and showed activity in a phase 1 trial [77]. In addition to the approved drugs mentioned above, other FLT3 inhibitors also have displayed clinical benefits. Published trials and their primary results are summarized in Table2. Sorafenib, already approved for renal cell cancer, thyroid cancer and hepatocellular carcinoma, were evaluated in either in a first-line and salvage situation combined with chemotherapy and HMAs (hypomethylating agents), showing promising results [78–84]. A novel second-generation FLT3 inhibitor crenolanib has shown possible benefits in combination with conventional chemotherapy, in either first-line and salvage treatment [85–87]. Lestaurtinib, however, failed to display clinical benefit when administered as maintenance therapy following standard treatment [88,89]. Biomedicines 2020, 8, 245 5 of 21

Table 2. Clinical results of FLT3 inhibitors.

SORAFENIB (BAY 43-9006) Sequential Authors and Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT Not Reached Rolling, et al. Lancet Oncol 2015 SORAML AML (age < 60) Newly diagnosed Sorafenib + Standard therapy II CR 60% (81/134) 21 mo. [9–32] 31% (42/132) (3-yr OS 63%) AML (age > 60) with 8.8 mo. (FLT3-ITD) 15.0 mo. (FLT3-ITD) Uy, et al. Blood Advances 2016 CALGB 11001 Newly diagnosed Sorafenib + Standard therapy II CR 74% (40/54) 53% (22/54) FLT3-ITD and/or TKD 7.8 mo. (FLT3-TKD) 16.2 mo. (FLT3-TKD) AML (age > 60) with CR/Cri 70% (19/27) 7.1 mo. 8.3 mo. Ohanian, et al. Am J Hematol 2018 Newly diagnosed Sorafenib + Azacitidine I/II 11% (3/27) − FLT3-ITD PR 7% (2/27) (only in responders) (in all participants) Sasaki, et al. Cancer 2019 AML with FLT3-ITD Newly diagnosed Soragenib + Standard therapy Retrospective CR/CRi 99% (78/79) 31 mo. [5.7–56.8] 17 mo. [11.1–22.4] 67% (53/79) − Muppidi, et al. Clinical Lymphoma AML with FLT3-ITD Newly diagnosed or relapsed Sorafenib + Decitabine Case Series CR/CRi 83% (5/6) Not Reported 5.1 mo. [1.9–14.5] 33% (2/6) Myeloma and Leukemia 2015 − Relapsed or refractory CR/Cri 43% (16/37) Ravandi, et al. Blood 2013 AML with FLT3-ITD Sorafenib + Azacitidine II 3.8 mo. [1.0–16.4] 6.2 mo. 16% (6/37) − (including prior allo-SCT) PR 3% (1/37) Bazarbachi, et al. Sorafenib as part AML with FLT3-ITD Relapsed ater allo-SCT Retrospective CR 39% (10/26) Not Reported (2-yr. OS 38%) 13% (3/26) Heamatologica 2019 − of/after salvage MIDOSTAURIN (PKC412) Sequential Authorsand Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT AML with FLT3-ITD Midostaurin + Standard Stone, et al. N Engl J Med 2017 RATIFY Newly diagnosed III CR 70% (504/717) 8.2 mo. [5.4–10.7] 74.7 mo. [31.5–inf.] 57% (287/504) and/or TKD induction/consolidation Midostaurin + Standard AML with FLT3-ITD Schlenk, et al. Blood 2019 AMLSG 16-10 Newly diagnosed induction/consolidation f/b II CR/CRi 76% (217/292) 13.2 mo. [10.0–18.3] 26.0 mo. [18.9–37.0] 62% (134/217) and/or TKD Midostaurin maitenance PR 1% (1/97) Relapsed or refractory or HI 46% (16/35 *) Fischer, et al. J Clin Oncol 2010 AML or high-risk MDS Midostaurin IIB Not Reported 4.3 mo. [3.5–5.2] 31% (42/132) − ineligible to standard therapy Blast 71% (25/35 *) * only in FLT3-mt Relapsed or refractory or CR/Cri 15% (8/54) 4.6 mo. [2.3–6.9] Strati, et al. Am J Hematol 2015 AML or high-risk MDS Midostaurin + Azacitidine I/II 5.1 mo. [3.5–6.7] 0% (0/8) − ineligible to standard therapy PR/MLFS 13% (7/54) * Duration of Response Walker, et al. Leukemia & Relapsed or refractory Midostaurin + Bortezomib + AML I CR/CRi 83% (19/23) Not Reported 10.8 mo. 63% (12/19) Lymphoma 2016 − (including prior allo-SCT) Chemotherapy(MEC) Maziarz, et al. Blood 2018 RADIUS AML with FLT3-ITD in 1st CR after allo-SCT Midostaurin + Standard of care II Not Applicable (18mo.-PFS 89%) Not Reported Not Applicable GILTERITINIB (ASP2215) Sequential Authors and Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT AML with FLT3-ITD CR/Cri 54% (134/247) Perl, et al. N Engl J Med 2019 ADMIRAL Relapsed or refractory Gilteritinib III 2.8 mo. [1.4–3.7] 9.3 mo. [7.7–10.7] 26% (63/247) and/or TKD PR 13% (33/247) AML with FLT3-ITD CR/Cri 41% (69/169) 4.6 mo. Perl, et al. Lancet Oncol 2017 Relapsed or refractory Gilteritinib I/II 7.1 mo. 22% (37/169) − and/or TKD PR 11% (19/169) * Duration of Response CR/Cri 60% (3/5 *) Usuki, et al. Cancer Science 2018 AML Relapsed or refractory Gilteritinib I PR 20% (1/5 *) Not Reported Not Reported Not Reported − * only in FLT3-mt. Biomedicines 2020, 8, 245 6 of 21

Table 2. Cont.

QUIZARTINIB (AC220) Sequential Authors and Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT Quizartinib + Standard Altman, et al. Blood 2018 AML Newly diagnosed induction/consolidation f/b I CR/CRi 74% (14/19) (Maximum 16.3 mo.) Not Reported 47% (9/19) − Quizartinib maitenance Cortes, et al. Blood 2019 QuANTUM-R AML with FLT3-ITD Relapsed or refractory Quizartinib III CR/Cri 48% (118/245) 1.4 mo. [0.0–1.9] 6.2 mo. [5.3–7.2] 32% (78/245) CR/Cri 47% (36/76) Cortes, et al. Blood 2018 AML with FLT3-ITD Relapsed or refractory Quizartinib IIB 12.3 mo. [9.7–16.1] 22.6 mo. [19.9–28.3] 37% (28/76) − PR 18% (14/76) CR/Cri 50% (125/248) 2.8 mo. [1.4–3.6] 5.8 mo. [4.9–6.8] * Cortes, et al. Lancet Oncol 2018 AML Relapsed or refractory Quizartinib II PR 25% (62/248) * duration of CR, 35% (61/176) − only in ITD-mt. * only in ITD-mt. only in ITD-mt. (0.4–22.8 mo.) Sandmaier, et al. Am J Hematol 2017 AML with FLT3-ITD in 1st CR after allo-SCT Quizartinib maintenance I Not Applicable (3.0–32.7 mo.) Not Applicable − * duration of maitenance CRENOLANIB (CP868596) Sequential Authors and Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT Crenolanib + Standard AML with FLT3-ITD Not Reached Wang, et al. Blood 2016 Newly diagnosed induction/consolidation f/b II CR/CRi 96% (24/25) Not Reported 50% (12/24) − and/or TKD (6 mo. OS 85%) Crenolanib maintenance CR/Cri 23% (3/13) AML with FLT3-ITD MLFS 8% (1/13) 3.0 mo. 12.7 mo. Randhawa, et al. Blood 2014 Relapsed or refractory Crenolanib II 26% (9/34) − and/or TKD HI 31% (4/13) * only in TKI-naïve * only in TKI-naïve * only in TKI-naïve AML with FLT3-ITD Crenolanib + Salvage chemotherapy Ohanian, et al. Blood 2016 Relapsed or refractory I CR/CRi 36% (4/11) Not Reported 8.5 mo. 75% (3/4) − and/or TKD (IDA/AraC) Iyer, et al. Blood 2016 AML Relapsed or refractory Crenolanib + Chemotherapy (HAM) I CR/CRi 67% (4/6) Not Reported Not Repoted 25% (1/4) − LESTAURTINIB (CEP701) Sequential Authors and Jounals Trial Name Objectives Disease Status Agents * Controls Not Shown Phase/Design Response Rate Median PFS Median OS allo-SCT AML with FLT3-ITD Slavage chemotherapy (MEC) f/b Levis, et al. Blood 2017 Relapsed II CR/CRi 26% (29/112) Not Reported 5.2 mo. 20% (22/112) − and/or TKD Lestaurtinib maintenance AML with FLT3-ITD Standard induction/consolidation f/b Knapper, et al. Blood 2017 Newly diagnosed III CR/CRi 92% (277/300) (5-yr. PFS 39–40%) (5-yr. OS 43–50%) 21% (58/277) − and/or TKD Lestaurtinib maintenance Biomedicines 2020, 8, 245 7 of 21 Biomedicines 2020, 8, x FOR PEER REVIEW 8 of 23

5. Mechanisms of Resistance to FLT3 Inhibitors 5. Mechanisms of Resistance to FLT3 Inhibitors 5.1. Primary Resistance 5.1. Primary Resistance Resistance to FLT3 inhibitors can be classified as primary resistance (innate resistance) and Resistance to FLT3 inhibitors can be classified as primary resistance (innate resistance) and secondary resistance (acquired resistance). In primary resistance, the effect of FLT3 inhibitors are secondary resistance (acquired resistance). In primary resistance, the effect of FLT3 inhibitors are prevented during the initial administration in an FL-dependent, FGF2-dependent, and stromal prevented during the initial administration in an FL-dependent, FGF2-dependent, and stromal CYP3A4-mediatedCYP3A4-mediated manner manner as as well well as asby the by activation the activation of other of signaling other signaling pathways pathways (Figure 1). (Figure Most 1). MostFLT3-mutant FLT3-mutant AML cells AML also cells express also wild-type express wild-type (WT) FLT3 (WT) concomitantly. FLT3 concomitantly. Since WT-FLT3 Since is sensitive WT-FLT3 isto sensitive FL and to is FL affected and is anegligiblyffected negligibly by FLT3 by inhibitors, FLT3 inhibitors, FL secretion FL secretion in the in thebone bone marrow marrow microenvironmentmicroenvironment leads leads toto thethe activationactivation of the FLT3/MAPK FLT3/MAPK pathway and and provides provides survival survival signals signals to AMLto AML cells cells during during induction induction and and consolidation consolidation therapy. therapy. Indeed, Indeed, certain certain studies studies have have demonstrated demonstrated that thethat co-existence the co-existence of WT-FLT3 of WT-FLT3 attenuated attenuated the anti-tumor the anti-tumor effects of effects FLT3 inhibitorsof FLT3 inhibitors on FLT3-mutated on FLT3- AML cellsmutatedin vitro AMLand cellsin in vivo vitro[88 and,90, 91in ].vivo In [88,90,91]. addition toIn FL,addition other to , FL, other cytokines, growth factors, growth and factors, soluble proteinsand soluble from proteins the bone from marrow the bo microenvironmentne marrow microenvironment have been studied have been with studied respect with to their respect resistance to againsttheir resistance quizartinib. against For quizartinib. example,fibroblast For example, growth fibroblast factor growth 2 (FGF2) factor induces 2 (FGF2) resistance induces byresistance activating FGFR1by activating and inducing FGFR1 and downstream inducing downstream MAPK signaling. MAPK FGF2 signaling. expression FGF2 expression in bone marrow in bone stromal marrow cells increasedstromal cells in patients increased with in FLT3-ITD-positivepatients with FLT3-I AMLTD-positive treated withAML quizartinib treated with and quizartinib was maximized and was prior tomaximized clinical relapse prior andto clinical the induction relapse of and resistance the induction mutations of resistance [92]. CXCL12, mutations a chemokine [92]. CXCL12, expressed a by osteoblastschemokine inexpressed the bone by marrow, osteoblasts is a ligand in the of bone CXCR4 marrow, expressed is a byligand hematopoietic of CXCR4 stemexpressed cells asby well ashematopoietic AML cells. Certain stem cells reports as well revealed as AML that cells. the Certain CXCR4 reports antagonist revealed plerixafor that the (AMD CXCR4 3100) antagonist selectively plerixafor (AMD 3100) selectively reduced the proliferation of FLT3-ITD AML blasts and increased reduced the proliferation of FLT3-ITD AML blasts and increased the sensitivity of FLT3-mutated the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of FLT3 inhibitors [93,94]; leukemic cells to the apoptogenic effects of FLT3 inhibitors [93,94]; therefore, the activation of the therefore, the activation of the CXCL12/CXCR4 axis may also induce resistance to FLT3 inhibitors in CXCL12/CXCR4 axis may also induce resistance to FLT3 inhibitors in AML cells. The inactivation of AML cells. The inactivation of TKIs by CYP3A4 is well established. In particular, hepatic CYP3A4 TKIs by CYP3A4 is well established. In particular, hepatic CYP3A4 inactivates all TKIs, including FLT3 inactivates all TKIs, including FLT3 inhibitors. Additionally, the expression of CYP3A4 in bone inhibitors.marrow stromal Additionally, cells attenuated the expression the activity of CYP3A4 of three in different bone marrow FLT3 inhibitors stromal cells (sorafenib, attenuated quizartinib, the activity ofand three gilteritinib) different FLT3in FLT3-ITD-positive inhibitors (sorafenib, AML quizartinib,[95]. and gilteritinib) in FLT3-ITD-positive AML [95].

Figure 1. Schematic mechanisms of primary resistance to FLT3 inhibitors. (1) Wild-type FLT3s are aFigure little sensitive 1. Schematic to FLT3 mechanisms inhibitors of primary and allow resistance downstream to FLT3 signaling inhibitors. by (1) binding Wild-type with FLT3s FLT3 are ligands. a (2)little FGF2 sensitive secreted to FLT3 from inhibitors bone marrow and allow stromal downst cellsream activates signaling FGFR1 by binding on leukemic with FLT3 cells whichligands. leads (2) to MAPKFGF2 secreted activation. from (3) bone Cell marrow adhesion stromal to the cells microenvironment activates FGFR1 may on leukemic also help cells leukemic which proliferation. leads to AntagonizingMAPK activation. CXCR4 (3) thatCell bindsadhesion to CXCL12 to the microenv on osteoblastsironment resulted may also in attenuated help leukemic leukemia proliferation. progression. (4) Upregulating CYP3A4 leads to the rapid inactivation of FLT3 inhibitors. Biomedicines 2020, 8, x FOR PEER REVIEW 9 of 23

BiomedicinesAntagonizing2020, 8, 245 CXCR4 that binds to CXCL12 on osteoblasts resulted in attenuated leukemia 8 of 21 progression. (4) Upregulating CYP3A4 leads to the rapid inactivation of FLT3 inhibitors.

5.2.5.2. Secondary Secondary Resistance Resistance due Due to to Addition Additionalal FLT3 FLT3 Mutations Mutations (on-Target (on-Target Resistance) Resistance) SecondarySecondary resistance resistance negates negates the the effects effects of of FLT3 FLT3 inhibitors inhibitors via via the the abnormalities abnormalities acquired acquired by by FLT3FLT3 inhibition, inhibition, such such as as additional additional mutations mutations in in FLT3 FLT3 (“on-target” (“on-target” resistance) resistance) and and defective defective factors factors apartapart from from FLT3 FLT3 (“off-target” (“off-target” resistance). resistance). Several Several genetic genetic mutations mutations associated associated with with FLT3 FLT3 inhibitor inhibitor resistanceresistance have have been been reported reported in in clinical clinical trials trials on on FLT3 FLT3 inhibitors. inhibitors. As As mentioned mentioned earlier, earlier, since since type type II inhibitorsII inhibitors originally originally have have no noaffinity affinity for for FLT3-TKD, FLT3-TKD, additional additional mutations mutations in in the the TK TK domain domain can can conferconfer resistance resistance via via the the elimination elimination of of the the inhibito inhibitoryry effect effect on on FLT3-ITD. FLT3-ITD. In In cases cases of of recurrence recurrence after after quizartinibquizartinib treatment treatment in in patients patients with with FLT3-ITD FLT3-ITD-positive-positive AML, AML, secondary secondary mutations mutations at at D835 D835 and and Y842Y842 residues residues as as well well as as at at the the commonly commonly known known “g “gatekeeperatekeeper residue” residue” F691 F691 in in the the kinase kinase domain domain havehave been been reported reported (Figure (Figure 1)1)[ [96].96]. InIn vitro, vitro ,Ba/F Ba/F33 cells cells expressing expressing FLT3-ITD FLT3-ITD and and one one additional additional TKD TKD mutation,mutation, detected detected in in patients patients with with clinical clinical resist resistanceance (+D835Y, (+D835Y, +D835V,+D835V, +Y842C,+Y842C, +Y842H,+Y842H, or or +F691L),+F691L), exhibitedexhibited resistance resistance to to the the growth growth inhibitory inhibitory effe effectct and and dephosphorylation dephosphorylation ac activitytivity of of quizartinib. quizartinib. TheseThese resistance mutationsmutations in in the the A-loop A-loop were were also also observed observed in patients in patients treated treated with sorafenib, with sorafenib, another anothertype II inhibitor.type II inhibitor. Furthermore, Furthermor duringe, the during treatment the treatment with gilteritinib with gilteritinib and crenolanib and crenolanib (a type I inhibitor), (a type Ithe inhibitor), additional the appearance additional of appearance FLT3-TKD mutations of FLT3-T inKD patients mutations with resistancein patients was with infrequent, resistance although was infrequent,the appearance although of F691L, the appearance a gatekeeper of mutation,F691L, a gatekeeper was observed mutation, (Figure was2). observed (Figure 2).

FigureFigure 2. 2. AdditionalAdditional FLT3 FLT3 tyrosine tyrosine kinase kinase domain domain mu mutationstations responsible responsible for for secondary secondary on-target on-target resistance.resistance. These These mutations mutations keep keep th thee TK TK domain domain in in active active FDG-in FDG-in form, form, not not allowing allowing the the type type II II inhibitorsinhibitors to to bind bind there. there. Mutation Mutationss in in a a“gate-keeping” “gate-keeping” residue residue F691 F691 shows shows the the universal universal resistance resistance to to both type I and II inhibitors. both type I and II inhibitors. Although the FLT3-F837K and FLT-C35S mutations occurred after the gilteritinib treatment in Although the FLT3-F837K and FLT-C35S mutations occurred after the gilteritinib treatment in one patient each, both were considered silent mutations as these did not induce self-proliferation one patient each, both were considered silent mutations as these did not induce self-proliferation in in Ba/F3 cells40. Among the 50 resistant patients treated with crenolanib, five FLT3 (D200N, K429F, Ba/F3 cells40. Among the 50 resistant patients treated with crenolanib, five FLT3 (D200N, K429F, Y572C, L601F, and F691L) mutations were observed in six patients; the D200N and L601F mutations Biomedicines 2020, 8, 245 9 of 21 did not result in leukemia [40]. Since the frequency of the acquired mutations in FLT3-ITD in patients with clinical resistance to quizartinib, a type II inhibitor, was 50% or less, other resistance mechanisms are also anticipated. In four out of eight patients treated with quizartinib, one or more resistance mutations were observed in the TK domain [96]. In addition to FLT3-ITD alleles, mutations in the TK domain of the original FLT3 allele were detected in seven individuals. Notably, the patients exhibited different frequencies of mutations between the original FLT3 allele and the FLT-ITD allele. In this study, the AML cells collected from one quizartinib-resistant patient did not acquire resistance mutations in either the original FLT3 allele or the FLT3-ITD allele. No mutations were detected in the genes apart from FLT3, although the existence of off-target resistance mechanisms was considered in this patient. These findings suggest the existence of a polyclonal resistance mechanism in patients with AML that relapses after quizartinib treatment.

5.3. Secondary Resistance Due to Non-FLT3 Abnormalities (off-Target Resistance) Resistant clones formed after treatment with gilteritinib and crenolanib, and a type I inhibitor that exerts an inhibitory effect on FLT3-TKD, have characteristics that are different from those observed after treatment with type II inhibitors. In a comparative genetic analysis before and after relapse in patients treated with gilteritinib, several distinct patterns of clonal selection were observed during the treatment period with gilteritinib [97]. In five out of 41 (12.2%) gilteritinib-resistant patients, FLT3 mutations were not observed in AML cells after the gilteritinib treatment; however, mutations in the RAS/MAPK pathway were present in all of the patients. These results suggest that mutant FLT3-negative clones acquire mutations in the RAS/MAPK pathway and expand as resistant clones. In 36 other patients, the resistant clones contained the original FLT3 mutation, and five of them acquired an F691L TKD mutation in addition to the original FLT3 mutation. In 10 out of the 36 patients with the original FLT3 mutation, additional mutations in the RAS/MAPK pathway, such as NRAS, KRAS, PTPN11, CBL, and BRAF mutations, were acquired. Of note, the mutations in the RAS/MAPK pathway and FLT-F691L mutations were mutually exclusive. In vitro experiments conducted in MOLM-14, an AML cell line with FLT3-ITD, where either mutant RAS or FLT3-F691L was transduced into the parental cells and gilteritinib was administered at low/high-dose (25 and 250 nmol/L), suggested that the RAS-mutant clones were more likely selected by the high concentration of the inhibitor, besides the FLT3-F691L which was more likely to be selected by a low one. Similar to RAS mutations [97,98], the activation of Axl-1, a member of the TAM family of receptor TKs, may also contribute to FLT3-resistance by constantly activating the RAS/MAPK and PI3K/Akt/mTOR pathway. Axl-1 was observed to be highly phosphorylated in midosutaurin-resistant AML cell lines and its resistance was diminished by the Axl-1 inhibition in vitro [99]. In another experiment, patient-derived AML cells with FLT3-ITD were co-cultured with stromal cells and treated with quizartinib [100]. The surviving cells underwent STAT5 activation, which consequently upregulated AXL, which was further enhanced by the hypoxic environment. Conversely, in patients eliciting poor response to crenolanib, several abnormalities have been observed in the loci encoding epigenetic regulators and granulocyte transcription factors, as well as in the cohesin complex. In particular, NRAS, STAG2, CEBPA, ASXL1, and IDH2 mutations were observed in FLT3-WT clones [40]. These findings suggest that the clones escaped and expanded during crenolanib therapy. However, TET2, IDH1, and TP53 mutations occurred simultaneously in FLT3-mutated clones during crenolanib treatment. These results suggest that the off-target resistance mechanism is more frequent when using type I inhibitors, such as gilteritinib or crenolanib, than type II inhibitors. Besides, IDH1 inhibitor [101] and IDH2 inhibitor [102,103], both approved by the FDA, are active against IDH1/2-mutant relapsed/refractory AML, though the significance of co-existing FLT3 mutations is not fully understood. In addition, the upregulation of the PI3K/AKT/mTOR pathway in resistant cell lines treated with sorafenib has also been reported [104]. Pim-1 is a proto- originally detected in hematopoietic cells that functions downstream of STAT5 [105]. Its overexpression induced resistance to lestaurtinib in BaF3/ITD cells and in samples collected from FLT3-ITD-positive patients [106]. Additionally, Pim kinase overexpression has been Biomedicines 2020, 8, 245 10 of 21

Biomedicines 2020, 8, x FOR PEER REVIEW 11 of 23 observed in the samples collected post sorafenib administration in patients with FLT3-ITD-positive AMLadministration compared to in the patients levels observedwith FLT3-ITD-positive in the samples collectedAML compared before administrationto the levels observed [107]. Pim-1 in the was associatedsamples withcollected an increased before administration expression of [107]. anti-apoptosis Pim-1 was proteins,associated such with as an Bcl-2, increased BCL-XL, expression and MCL-1, of inanti-apoptosis FLT3 inhibitor-resistant proteins, such cases as Bcl-2, [25,108 BCL-XL,–110]. Inand particular, MCL-1, in theFLT3 observed inhibitor-resistant resistance cases may [25,108– be partly induced110]. In by particular, Pim-1. O theff-target observed abnormalities resistance may along be with partly FLT3 induced signaling by Pim-1. are schematically Off-target abnormalities summarized in Figurealong with3. FLT3 signaling are schematically summarized in Figure 3.

FLT3 AXL Axl-1

Cytoplasm NRAS KRAS BRAF RAS PI3K JAK CBL PTPN11

MEK Akt STAT5 PIM1

ERK mTOR Pim-1

ASXL1 TET2 Survival/Proliferation DNMT3A STAG2 IDH1 Epigenetic/ Isocitrate IDH2 Transcriptional metabolism TP53

Nucleus Mitochondria

Figure 3. Schematic description of genetic abnormalities (mutations or upregulation) associated with secondaryFigure 3. oSchematicff-target resistance description to of FLT3 geneti inhibitors.c abnormalities Mutations (mutations involved or upregulation) in the RAS /associatedMAPK pathway with weresecondary reported. off-targetNRAS resistancemutation to is theFLT3 most inhibito commonrs. Mutations among involved them. Axl-1, in the coded RAS/MAPK by the pathwayAXL gene, is awere receptor reported. tyrosine NRAS kinase mutation that leadsis the tomost the common activation among of RAS them./MARK Axl-1, and coded PI3K by/Akt the/ mTORAXL gene, pathway. is a Thereceptor upregulation tyrosine of thekinaseAXL thatgene leads was to observed the activation in midostaurin-resistant of RAS/MARK and AML PI3K/Akt/mTOR cell lines. Pim-1 pathway. is part of The upregulation of the AXL gene was observed in midostaurin-resistant AML cell lines. Pim-1 is part the downstream signaling of STAT5, contributing cell survival and proliferation as well as cell migration. of the downstream signaling of STAT5, contributing cell survival and proliferation as well as cell A lestaurtinib-resistant AML cell line showed the overexpression of Pim-1. Other gene mutations migration. A lestaurtinib-resistant AML cell line showed the overexpression of Pim-1. Other gene commonly seen in AML regardless of FLT3 status were also detected. Although a direct relationship mutations commonly seen in AML regardless of FLT3 status were also detected. Although a direct with FLT3 signaling was not suggested, these mutations have an essential role in maintaining leukemic relationship with FLT3 signaling was not suggested, these mutations have an essential role in clones by modulating epigenetic/transcriptional regulations (e.g., ASXL1, TET2, DNMT3A and STAG2), maintaining leukemic clones by modulating epigenetic/transcriptional regulations (e.g., ASXL1, altering the metabolism of the citrate acid cycle (e.g., IDH1 and IDH2) and preventing apoptosis TET2, DNMT3A and STAG2), altering the metabolism of the citrate acid cycle (e.g., IDH1 and IDH2) (e.g., TP53). and preventing apoptosis (e.g., TP53). Biomedicines 2020, 8, 245 11 of 21

6. Strategies to Overcome Resistance to FLT3 Inhibitors

6.1. Development of Novel Agents Previous reports suggest that on-target resistance tends to occur in patients after type II inhibitor treatment, while off-target resistance is likely to occur after type I inhibitor treatment. Since these reports are currently limited to patients recruited during clinical trials, for a better understanding of the mechanism underlying the resistance to each FLT3 inhibitor, it is necessary to determine the characteristics of patients with resistance in real-word settings. In addition, to counter the gatekeeper mutation (F691L) in FLT3, which confers resistance to all existing FLT3 inhibitors, it is necessary to develop a novel FLT3 inhibitor. As described above, while type I inhibitors can also inhibit FLT3-TKD, they exhibit low selectivity, whereas although type II inhibitors cannot inhibit FLT3-TKD, they exhibit high selectivity. FLT3-TKD inhibitory activity and FLT3 selectivity share a trade-off relationship. To resolve these issues, a novel FLT inhibitor known as FF-10101 was designed, which would form covalent bonds with the C695 residues of FLT3. The creation of covalent bonds by FF-10101 enables the selective and irreversible inhibition of FLT3 in either the active or the inactive form [111]. Furthermore, the unique binding method of FF-10101 exerts wide inhibitory action against various FLT3 mutations, including F691L. Currently, phase 1/2 trials are underway to evaluate its safety, tolerability, pharmacokinetics, and efficacy against recurrent refractory AML (NCT03194685). In addition, several agents that may overcome or prevent resistance are currently under investigation. A pan-PIM/FLT3 inhibitor SEL24 [112], a type II FLT3 inhibitor MZH29 [113], a MERTK/FLT3 inhibitor MRX-2843 [114], a BCR-ABL inhibitor [115], and a multiple tyrosine kinase inhibitor [116] have exhibited anti-tumor activity in cases with FLT3-TKD, including those with the F691 pointmutation.

6.2. Combination with Different Class Agents Existing FLT3 inhibitors are now being tested in combination with HMAs, standard chemotherapy, bortezomib (proteasome inhibitor), atezolizumab (anti-PD-L1 antibody), (BCL-2 inhibitor), milademetan (MDM2 inhibitor) and homoharringtonine (STAT inhibitor). Ongoing trails of combination strategy are summarized in Table3 Preclinically, FLT3 ligand-mediated resistance was attenuated by the dual inhibition of AKT/FLT3 in vivo [117]. The combination of the MEK and FLT3 inhibitors as well as the dual inhibition of MEK/FLT3 proved to be effective against resistance-conferring FLT3 mutations in in vivo and in vitro mutations [97,118]. The sensitization of FLT3 inhibitors can serve as an alternate strategy. Proteasome inhibitors, arsenic trioxide (ATO), and a CDK4/6 inhibitor downregulated FLT3 molecules in FLT3-ITD AML cells by promoting cytotoxic autophagy, inhibiting the expression of FLT3 RNAs, and dysregulating the transcription of FLT3 and PIM1, respectively [119–121]. The inactivation of ATM or its downstream effector G6PD also induced synthetic lethality along with FLT3 inhibition by enhancing mitochondrial oxidative stress, which eventually resulted in tumor apoptosis [122]. Biomedicines 2020, 8, 245 12 of 21

Table 3. Clinical trials of FLT3 inhibitors.

SORAFENIB (BAY 43-9006) Trial Number Objectives Disease Status Agents * Controls Not Shown Phase/Design AML with FLT3-ITD NCT01371981 Newly diagnosed Sorafenib + Bertezomib III (high allelic ratio) Sorafenib + Homoharringtonine NCT03170895 AML with FLT3-ITD Newly diagnosed or relapsed II (STAT inhibitor) MIDOSTAURIN (PKC412) NCT03686345 Core binding factor AML Newly diagnosed Midostaurin + Standard induction II Midostaurin + Standard AML with FLT3-ITD NCT03280030 Newly diagnosed induction/consolidation f/b II and/or TKD Midostaurin maitenance Midostaurin + Standard AML with FLT3-ITD NCT03512197 Newly diagnosed induction/consolidation f/b III and/or TKD Midostaurin maitenance Midostaurin + Standard AML with FLT3-ITD NCT03379727 Newly diagnosed induction/consolidation f/b III and/or TKD Midostaurin maitenance GILTERITINIB (ASP2215) Gilteritinib + Standard NCT02236013 AML Newly diagnosed I induction/consolidation AML with FLT3-ITD Newly diagnosed and NCT02752035 Gilteritinib + Azacitidine III and/or TKD ineligible to standard therapy AML with FLT3-ITD Gilteritinib + Atezolizumab NCT03730012 Relapsed or refractory I/II and/or TKD (anti-PD-L1 antibody) Gilteritinib + Standard NCT02310321 AML Newly diagnosed I/II induction/consolidation AML with FLT3-ITD Gilteritinib + Salvage NCT03182244 Relapsed or refractory III and/or TKD chemotherapy QUIZARTINIB (AC220) Quizartinib + Standard NCT02668653 AML with FLT3-ITD Newly diagnosed III induction/consolidation Quizartinib + Standard NCT03723681 AML Newly diagnosed I induction/consolidation Quizartinib + Standard NCT02834390 AML Newly diagnosed IB induction/consolidation Relapsed or refractory or Quizartinib + Milademetan NCT03552029 AML with FLT3-ITD I ineligible to standard therapy (MDM2 inhibitor) Quizartinib + Homoharringtonine NCT03135054 AML with FLT3-ITD Newly diagnosed or relapsed II (STAT inihibitor) Quizartinib + Decitabine + NCT03661307 AML with FLT3-ITD Newly diagnosed or relapsed I/II Venetoclax (BCL-2 inhibitor) Quizartinib + Venetoclax NCT03735875 AML with FLT3-ITD Relapsed or refractory IB/II (BCL-2 inhibitor) CRENOLANIB (CP868596) AML with FLT3-ITD Crenolanib + Standard NCT03258931 Newly diagnosed III and/or TKD induction/consolidation AML with FLT3-ITD NCT02283177 Newly diagnosed Crenolanib + Standard induction II and/or TKD AML with FLT3-ITD Crenolanib + Salvage NCT03250338 Relapsed or refractory III and/or TKD chemotherapy AML with FLT3-ITD Crenolanib + Salvage chemo. or NCT02400281 Relapsed or refractory I/II and/or TKD Azacitidine Crenolanib + Salvage NCT02626338 AML Relapsed or refractory I/II chemotherapy AML with FLT3-ITD Crenolanib + Standard NCT01522469 Relapsed or refractory IB and/or TKD induction/consolidation OTHERS AML with FLT3-ITD NCT00783653 Newly diagnosed Sunitinib + Standard induction I/II and/or TKD AML with FLT3-ITD Lestaurtinib + Salvage NCT00469859 Relapsed or refractory I/II and/or TKD chemotherapy Biomedicines 2020, 8, 245 13 of 21

6.3. Genetic Mutation Analysis As described, the presence or absence of mutations in FLT3 has become an important determinant of the treatment methods in AML. Currently, a companion diagnostic tool LeukoStratCDx (Invivoscribe, Inc., San Diego, CA, USA) is widely used for the clinical use of FLT3 inhibitors in Japan, the United States, and Europe, among others. However, LeukoStratCDx is only able to detect D835 and I836 mutations and cannot detect any other FLT-TKD mutations, including F691L. Therefore, the instrument might incorrectly analyze the condition in patients with FLT3-TKD that is potentially treatable by FLT3 inhibitors. Although intensive chemotherapy has ensured substantial clinical benefit in AML patients, several patients eventually require , particularly young patients. In addition, CEBPA and NPM1, and recently TP53, ASXL1 and RUNX1, have been determined to be important markers prognosis [27,31,123], transplant eligibility, and treatment strategy. Even after FLT3-ITD/TKD becomes undetectable in remission, the expression of persistently mutated genes such as DNMT3A, TET2, SRSF2, and ASXL1 continues to be associated with high relapse rates and poor prognosis [124]. Although the negative prognostic impact of FLT3-ITD might be, at least partially, attenuated by upfront haploidentical stem cell transplantation (haplo-SCT) [125], FLT3 inhibitors remain one of the useful choices for treating the majority of FLT3-mutated AML patients, especially elderly and/or unfit people. To overcome resistance to FLT3 inhibitors, mutation analyses in patients with resistance to FLT3 inhibitors are required to identify the genetic abnormalities that contribute to drug-resistance and determine additional therapeutic targets. Genome-wide analysis using the CRISPR-Cas9 single-guide RNA (sgRNA) library, a vector-mediated technique for the knockdown of particular genes, revealed that the loss of SPRY3 and GSK3 confers resistance to quizartinib by inducing the reactivation of the FGF/RAS/ERK pathway and Wnt signaling [126]. Likewise, in addition to FLT3 mutations, it is necessary to comprehensively evaluate various genetic abnormalities; comprehensive mutation testing by next-generation sequencing (NGS) is expected to enable this. Accordingly, we analyzed the cancer-related genetic abnormalities (i.e., in an NGS panel) in patients with AML who were ineligible for intensive chemotherapy or developed recurrent/refractory cancer after initial therapy (Foundation One Heme; we planned HM-SCREEN-JAPAN, an observational study that analyzes and evaluates the relationship between prognosis by F1H). The primary goal of this project is the development of F1H and the promotion of targeted therapy for AML [127].

7. Conclusions and Future Perspectives This paper described the principal mechanisms of resistance to FLT3 inhibition and the current investigations to overcome it. Secondary on-target mutations (i.e., FLT3-TKD) can be managed by choosing type I inhibitors such as gilteritinib that are consistently active against FLT3-TKD as well as FLT3-ITD, except for a “gatekeeper” F691L mutation. Covalently-coupling FF-10101 and other novel FLT3 inhibitors are now under investigation and have shown promising data on FLT3 F691L. Strategies for secondary off-target abnormalities and a part of primary resistance cannot be simple, regarding the diverse relating genomic abnormalities and complex patterns of clonal evolution. Nevertheless, some genetic abnormalities are/will be clinically targetable, expecting a synergistic anti-tumor effect with FLT3 inhibition. For example, several agents targeting BCL-2, MDM2 and STAT as well as conventional chemotherapy are being evaluated in combination with FLT3 inhibitors. Similarly, abnormal RAS and PIM1 pathways as well as metabolic modifications (e.g., G6PD inactivation) are subject to preclinical investigations. Recent studies have suggested the non-negligible importance of clone-evolutional patterns in terms of acquiring resistance, which possibly affects clinical strategy in managing FLT3-mutated AML. Simply, when you find two distinct targetable mutations and the corresponding agents are available (e.g., FLT3-ITD and IDH2 mutation), you can choose either one agent if both mutations are limited in a single leukemic clone, but if each mutations are found in different clones, it is worth considering combination or sequential therapy, if allowed. Routine and successive gene sequencing will help detecting additional targetable mutations and determining Biomedicines 2020, 8, 245 14 of 21 individual patterns of clone evolution, which would improve our clinical approaches along with the development of novel agents and combination strategies. Several new agents such as FLT3 inhibitors can create overlapping treatment options, especially in the elderly, unfit AML patients as well as in relapse/refractory AML patients. A lot of clinical trials evaluating the efficacy of promising investigational drugs in AML are ongoing and more drugs will go to the market than ever before. Based on the resistant mechanisms during treatment, how to use these new agents properly is one of the issues with the treatment of AML. Physicians should select an optimal treatment depending on factors such as age, performance status, comorbidities, and genome profiling analysis upon new diagnoses and during treatment.

Author Contributions: M.E. and S.C. wrote the first draft and all authors revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This paper was supported by the National Cancer Research & Development expenses grant. Conflicts of Interest: Y.M. received research funding from Ono and honoraria from Bristol-Myers Squibb, Novartis, and Pfizer. The other authors declare no conflict of interest.

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